Seed weight or seed size is an important agronomic trait in Brassica crop species.
For several crop species, Quantitative Trait Loci (QTLs) have been mapped for seed size and weight. In Brassica, several QTLs for seed weight have been mapped (Cai et al., 2012, BMC Genetics 13: 105; Zhang et al., 2011, Theor Appl Genet 122:21; Fan et al., 2010, Theor Appl Genet 121: 1289). Two candidate genes, BnMINI3a and BnTTG2a have, based on QTL mapping, been assigned as candidate genes (Fan et al., 2010, supra). BnMINI3a and BnTTG2a are Brassica homologs of WRKY transcription factors of Arabidopsis MINI3 and TTG2, respectively. Cai et al., 2012 (supra) mapped location of several candidate yield genes to yield QTLs and found localization of Arabidopsis genes TTG2 and GS5 close to yield QTL TSWA1; GW2 at the same position of TSWA2; CKI1 and MN1 nearby the peak of TSWA4; MINI3 in the confidence interval of TSWA5b; FIE in the confidence interval of TSWA5a; AHP3, AHP5 and MEA in the same confidence interval of TSWA10; and AGL62, GS3 and GASA4 on the peaks or in the confidence intervals of TSWC2a, TSWC2b, and TSWC2c, respectively. For two major QTLs, TSWA7a and TSWA7b, no known information about candidate genes could be inferred from the map.
In addition to the above genes, several other genes involved in seed size or weight regulation have been identified (see Cai et al., 2012 (supra) for an overview). It has further been described that Brassica plants overexpressing different variants of the REV gene have increased seed size and thousand seed weight (WO2007/079353, US2011/0271405). Brassica genes which increase seed weight when overexpressed in Arabidopsis are AOX1 (WO2012/100682) and a wri1-like gene (Liu et al., 2010, Plant Physiol Biochem 48: 9). Other genes affecting seed size or weight are MNT (WO2005/085453), Cytokinin Oxidase (US2005/0150012), CYP78A7 (US2010/0281576), sucrose isomerase (WO2012/119152), Polycomb group genes (WO2001/038551), Giberrelin 20-oxidase (US2009/0007295), and sorbitol dehydrogenase (WO2008/144653). In US2011/0265225, four rice genes have been described that increase seed size when overexpressed in rice. WO2003/096797 describes overexpression of several genes from several plant species and the effect on seed size and weight in Arabidopsis and soybeans.
Li et al (2008, Genes Dev 22:1331) and WO2009/047525 describe Arabidopsis DA1, encoding a predicted ubiquitin receptor, which sets final seed and organ size by restricting the period of cell proliferation. A da1-1 mutant has been identified in genetic screens in Arabidopsis thaliana that increases both seed and organ size. The increased seed mass was observed only when maternal plants were homozygous for the da1-1 mutation. The da1-1 mutant contains a single-nucleotide G to A transition in gene At1g19270, predicted to cause an arginine-to-lysine change in a conserved amino acid at position 358. The da1-1 phenotypes were complemented by the wild-type DA1 gene and by transgenic expression of a wild-type DA1 full length cDNA. Disruption of the DA1 gene did not cause an obvious growth phenotype. Lines heterozygous for the da1-1 mutation had a seed and organ size nearly similar to the wild type, whereas plants with the da1-1 mutation combined with the da1 knock-out allele displayed a similar phenotype to da1-1.
Two Brassica rapa DA1 orthologs, BrDA1a and BrDA1b have been identified (WO2009/047525). The amino acid sequence of BrDA1a is more close to the Arabidopsis DA1 (AtDA1) amino acid sequence, but BrDA1b was predicted to have more similar biochemical features to AtDA1. Transgenic Arabidopsis da1-1 plants expressing 35S-BrDA1a showed at least partial complementation of the da1-1 phenotype, whereas 35S-BrDA1b transgenic plants showed full complementation of the da1-1 phenotype. When the BrDA1a cDNA with a mutation equivalent to the R358K mutation of the Arabidopsis DA1 (BrDA1aR358K) was overexpressed in wild-type Arabidopsis, typical da1-1 phenotypes were observed.
Cai et al, 2012 (supra) describe that Brassica rapa and Brassica oleracea each contain two copies of the DA1 gene. The homologous DA1 genes were positioned on a B. napus linkage map and aligned with Thousand Seed Weight (TSW) QTL loci. Whereas several candidate yield genes were linked to TSW QTLs by Cai et al. (see above), DA1 did not colocalize with one of the 11 tested TSW QTLs.
In order to use the DA1 gene to increase seed yield in Brassica, a need remains to understand the relative contribution of the different DA1 genes to seed weight. The isolation of mutant alleles corresponding to da1 in economically important Brassicaceae plants, such as oilseed rape, may be complicated by the amphidiploidy in oilseed rape and the consequent functional redundancy of the corresponding genes.
Thus, the prior art is deficient in teaching the identity of DA1 genes in amphidiploid Brassica species, as well as the contribution of the different DA1 genes to seed weight. As described hereinafter, this problem has been solved, allowing to modify DA1 with the aim to increase seed weight in Brassica, as will become apparent from the different embodiments and the claims.